Al Leydecker; 15 April 2006: page 1 of 4
Methods (UCSB-LTER Laboratories)
Water sampling and chemical analyses
Stream water samples were collected manually at mid-depth near the center of flow. Sample bottles (and caps) of high-density polyethylene (HDPE) were rinsed three times with deionized water before being used, and twice with sample water immediately prior to being filled; samples are placed in coolers as soon as possible and are transported on ice. Once in the laboratory, samples were stored at 4° C.
Samples for dissolved constituents are generally filtered in the field through Gelman A/E glass fiber filters, pre-flushed with deionized and sample water. A syringe is used to force the sample thru the filter unit. Stormflow samples with high sediment concentrations cannot be field-filtered and may be centrifuged or allowed to settle before filtration in the laboratory.
Nutrient analysis
Samples were analyzed for nitrogen (dissolved organic nitrogen, nitrate (NO3 + NO2) and ammonium) and phosphorus (soluble reactive phosphate, i.e., SRP). Nitrate, ammonium and phosphate were determined colorimetrically on a Lachat® auto-analyzer. Ammonium was measured by adding base to the sample stream converting ammonium to ammonia, which diffuses across a Teflon® membrane (Willason and Johnson, 1986) and into phenol red pH indicator. Nitrate was measured using a standard Griess-Ilosvay reaction after Cd reduction (EPA, 1983). Phosphate was measured after reaction with ammonium molybdate and antimony potassium tartrate and reduction by ascorbic acid with heating at 45° C.
Detection limits were 0.3 µmol L-1 for NH4+ and PO43- and 0.5 µmol L-1 for NO3-; accuracy is ±5%. Total dissolved nitrogen (TDN) was determined after persulfate digestion (Valderrama, 1980) followed by measurement of nitrate. The basic persulfate reagent was added to a separate aliquot at the time of initial processing or laboratory filtration and the digestion completed within one week Detection limit was 0.5 µmol L-1 and accuracy + 10%. Dissolved organic nitrogen (DON) was computed as the difference between TDN and dissolved inorganic nitrogen (DIN: nitrate and ammonium).
The goal was to analyze inorganic nutrient samples and begin the digestion of total dissolved nitrogen samples within 48 hours of collection, and we were able to meet this goal for most of the samples collected. However, during winter storm periods, when high sediment concentrations prevented filtration in the field and the laboratory was inundated with hundreds of samples, the 48 hour limit was often exceeded by 1 to 5 days. To evaluate the effect of delay, three types of samples were collected from six streams with widely varying nutrient chemistry: (1) samples filtered in the field and analyzed in duplicate within 12 hours; (2) samples filtered in the laboratory on the day of collection, stored at 4° C, and repeatedly re-analyzed after delays of 1 to 14 days; and (3) an unfiltered sample, stored at 4° C, sub-samples of which were repeatedly filtered and analyzed after similar delays. Numerous duplicate and deionized water samples provided quality assessment and control. The average error (the combined error of processing, delay, instrument calibration and analysis) for nitrate was 5 to 10 % (the higher percentage error in the second week of delay), 10 % for phosphate, and 20 % for ammonium. Samples filtered within two days showed almost no variation in nitrate and phosphate from initial values, while ammonium was usually within 10 %. Delays greater than 2 days did sometimes cause significant increases in ammonium concentrations.
Problems with nutrient results
Error and imprecision are part of all laboratory analysis; a result is never simply a number, it’s a number plus or minus some error. Total nitrogen and total phosphorus are analyzed to determine the concentrations of organic nitrogen and organic phosphorus in a sample. The inorganic concentration is simply subtracted from the total – phosphate from total phosphorus, inorganic nitrogen (nitrate + ammonium) from total nitrogen – what remains is the organic fraction.
Sometimes analysis error or the precision of the result are such that the inorganic concentration is higher than the total concentration, i.e., a larger number has to be subtracted from a smaller. For example, the total phosphorus concentration may end up being lower than the phosphate in a sample. Obviously, this cannot be true; something either went wrong or the precision of the analysis was not high enough to produce a satisfactory result by subtraction. [Consider a ±5 % error in both analyses and think of the phosphate result being 5 % too high while the total phosphorus concentration is 5 % too low.] It happens about 4 % of the time with nitrogen (acceptable, particularly when concentrations are high), but 50 % of the time with phosphorus. The phosphorus results present a real problem, one that the UCSB laboratory has not been able to solve. Something in local stream water removes phosphorus from solution during the test procedure and since the total phosphorus results are undependable, phosphate is the more reliable indicator.
This is not an important distinction. Phosphate makes up a large majority of total phosphorus in Channelkeeper samples, and nitrate is the dominant nitrogen fraction at most sites. Analysis of filtered vs. unfiltered samples to determine nutrient composition is another difference without a distinction. Tests on filtered and unfiltered samples at most of the Ventura sites show no statistical difference between these two types of samples. Except for those rare rainy days, Venturaand Goletawater is relatively sediment free.
Particulate analysis
Samples for particulate analysis were collected in separate 2 liter polypropylene bottles. Particulates are obtained by filtering measured volumes of sample water through 25mm Gelman A/E 1-micron filters. Two filters, one for carbon, hydrogen and nitrogen analysis (CHN), the other for phosphorus, were obtained. While filtering, sample bottles were well shaken then rapidly pipetted or poured into a graduate cylinder cut off at an appropriate volume (e.g., 25, 50, 100 ml); volumes wereselected so that particulates significantly darkened the filter without creating a sediment cake.
Particulate carbon and nitrogen were measured by combustion of filtered samples using a Control Equipment Corp (now Exeter Analytical) CHN analyzer (MSI Analytical Lab), operated under the manufacturer’s recommended conditions. Results are provided initially in micrograms of the element, and converted to umoles/L by dividing by the atomic weight and by the volume of sample filtered. Instrument precision is +/- 1 ug, and detection limits are about 2ug. Actual detection limits, however, depend on the variability of filter blanks, which typically run about +/- 5ug. Measurement accuracy was monitored by analysis of one or more control samples (pure organic compounds of known composition) with each sample batch, and is better than +/- 0.3%, or +/- 3ug, whichever is greater. QA/QC proceduresinclude periodic re-analysis of calibration standards during each sample batch run, and post-run evaluation or results for validity.
Particulate phosphorus concentrations were obtained by dry combustion (550oC for 2 hours) of the filter, followed by a digestion with boiling in HCl (Melack Laboratory). Digested samples are stored for up to 4 months in 14 ml screw cap plastic vials. Digests are neutralized with NaOH and afterwards the digest is assayed for PO43-. Detection limit is about 1.0 µM and accuracy is ± 10%.
Sediment analysis
Samples for the gravimetric determination of suspended solids were collected in separate polypropylene bottles. A known volume of well mixed sample is filtered through a tarred 47mm Gelman A/E filter. After drying at 105oC for 2 hours, the weight of the filter plus residue is obtained (Melack Laboratory). Typical standard deviation of the test methodology is 5.3 g/L, Method B (ASTM-D 3977-97, 1997).
Miscellaneous analyses
Specific conductance (or electrical conductivity) of unfiltered water is measured on all samples with a conductivity bridge (cell constant = 0.1) and readings corrected to 25° C; a circa 1400 µS/cm standard is used for daily calibration. Gran titrations for ANC and pH measurements are done on specifically collected unfiltered samples using a digital pH meter and a Ross Orion combination electrode (Wetzel et al., 1979). On occasion filtered samples are used for ion analysis: chloride, nitrate and sulfate by ion chromatography (Dionex model 2010i or DX500); calcium, magnesium, sodium and potassium by flame atomic-adsorption spectroscopy (Varian model AA6 or Spectraa 400); and silica colormetrically (molybdo-silicate method; (Strickland et al., 1972)) on a Lachat Auto Analyzer (detection limit 0.5 µM). The average analytical error for anion and cation concentrations above 10 µeq/L is3 %, 5% for concentrations below 2 µeq/L (Melack Laboratory).
Lachat Standards and QA/QC
The Lachat AE Ion Analyzer is set up with a pump rate setting at 50 rpm and a cycle period 50 sec. All reagents and standards are made with polished MilliQ water (12-18 Mohm). Instrument calibration standards are made fresh every 2 days from 5 mM mixed standard (NH4+, NO3- and PO43-). Calibrations are performed twice before analyzing samples. Slopes needed to be within 10-20% of values obtained under similar analytical conditions, and within 5% of each other before samples are analyzed. Three blanks are obtained by flushing MilliQ (or comparable) system for 2 minutes and then filling 3 lachat tubes. Blanks are run after every calibration, and the average is deducted from analyzed values.
A calibration standard is reanalyzed after every 10 samples (“check standard”) and the instrument is recalibrated if it drifted beyond 5% of calibration value. Sometimes recalibration is not practical and sample values are corrected post-run if check standard is beyond 5%. Detection limit is about 0.3 µM for NH4+ and PO43-, about 0.5 µM for NO3-, and accuracy is about ±5%. Samples off scale for any channel are diluted with DI water and reanalyzed.
In addition to fresh instrument standards, QC standards mixed from independent primary standards are analyzed most days. These primary standards are also NIST-traceable or mixed directly from ACS-grade crystals. Accuracy between standards is generally within 10%, and sample values are not corrected for differences.
REFERENCES
ASTM-D 3977-97. 1997. Standard test methods for determining sediment concentration in water samples. Annual Book of ASTM Standards. 11.01.
Grasshoff, K. 1976. Methods of Seawater Analysis, Verlag Chemie, Lachat Instruments, Inc.
Lachat Instruments, Inc. 2000. Analytical methods. Milwaukee.
SBC-LTER. 2003. Santa Barbara Coastal Long Term Ecological Research Project. University of California, Santa Barbara,
Strickland, J. D. and T. R. Parsons. 1972. A practical handbook of seawater analysis. Bulletin of Fisheries Research Board of Canada(167): 310.
USEPA. 1983. Nitrogen, Nitrate-Nitrite. Method 353.2 (Colorimetric, Automated, Cadmium Reduction). Methods for Chemical Analysis of Water and Wastes(EPA-600/ 4-79-020): 353-2.1 -- 353-2.5.
Valderrama, J. C. 1980. The simultaneous analysis of total nitrogen and total phosphorus in natural waters. Marine Chemistry, 10: 109-122.
Wetzel, R. and G. Likens. 1979. Limnological Analyses. Philadelphia, W.B. Saunders: 357.
Willason, S. W. and K. S. Johnson. 1986. A rapid, highly sensitive technique for the determination of ammonia in seawater. Marine Biology, 91: 285-290.